Bone Fracture Risk and Renal Dysfunction in a Highly Cadmium Exposed Thai Population

Background: Paddy fields in the Mae Sot, Tak Province of Thailand are polluted with unsafe levels of cadmium (Cd). Elderly populations have a high Cd body burden, putting them at elevated risk of renal dysfunction and bone fractures. We aimed to compare bone fracture risk between glomerular dysfunction, proximal tubular dysfunction, and calcium (Ca) handling abnormalities. Study design: A cross-sectional study. Methods: Serum osteocalcin and cross-linked N-telopeptide of type I collagen were used to detect bone metabolism abnormalities, whereas glomerular filtration rate, serum cystatin C, urinary β2-microglobulin (β2-MG) and fractional excretion of calcium (FECa) were used to indicate renal dysfunction. Urinary Cd was used as an exposure marker. Results: FECa >2% was associated with high bone fracture risk in both genders. The adjusted odds of bone fracture risk were 6.029 and 3.288 in men and women, respectively with FECa >2% relative to the FECa <2% group. Proximal tubular dysfunction and glomerular dysfunction did not significantly relate to the risk of bone fracture. Conclusions: Abnormal Ca handling is a key risk factor for bone fracture in Cd-exposed people. Men and women were at risk of bone fracture risk at a similar rate. FECa was a specific indicator of Ca wasting and was more cost-effective compared to β2-MG and serum cystatin C. We recommend using FECa to monitor abnormal Ca metabolism in individuals with FECa>2%. Reduced renal toxicant exposure and Ca supplementation are recommended for Cd-exposed populations to reduce bone fracture risk.


Introduction
igh levels of cadmium (Cd) are exposure causes renal and bone abnormalities. Itai-itai disease is the most severe pathology caused by high-level cadmium exposure 1 . At low Cd exposure levels, exposed populations are also at risk of bone disease. The prevalence of osteoporosis in a Chinese Cd-exposed population was 2.09 times higher than reference group 2 , and bone fracture risk in an exposed population from Sweden was 8.80 times higher than a reference population 3 .
Cadmium contamination was identified in the Mae Sot district in Thailand, where a zinc mine operated. A survey of Cd levels in rice paddy soil showed that Cd in the polluted area ranged between 0.5-284 mg/kg, which was higher than in other areas of Thailand (0.01-1.30 mg/kg) 4 . In addition, 95% of surveyed rice grain samples were contaminated with Cd levels greater than 0.1 mg/kg, the level considered safe 5 . Rice is a staple food for the population of Mae Sot, putting them at increased risk of Cd toxicity 6 . There were 7697 inhabitants in the contaminated area and 7.2% of those inhabitants showed urinary Cd (U-Cd) >5 µg/g Cr 6 , a level associated with increased proximal tubular dysfunction 7 . A follow-up survey in 2007 found a 19.9% prevalence of permanent proximal tubular dysfunction and a 16.9% prevalence of glomerular dysfunction among the Cd-exposed population, which was higher than an unexposed Thai population 8 . Their exposure level was also greater than the threshold level for osteoporosis and increased bone fracture risk 9 .
Biomarkers of bone metabolism status have the advantage of identifying changes in bone metabolism and physiology earlier than measurements of bone mineral density 10 . Bone biomarker determination is a non-invasive and comparatively inexpensive tool to assess metabolic bone disease. Serum osteocalcin (OC) and urine cross-linked N-telopeptide of type I collagen (NTx) levels have previously been associated with an increased risk of bone fracture in clinical and epidemiological studies 11 . In a Cd-polluted area in Japan, associations between U-Cd and NTx and Cd-induced osteoporosis gradually developed in exposed subjects after cessation of exposure 12 . A proposed explanation for Cdinduced bone disease is kidney dysfunction, particularly proximal tubular abnormalities 13 . However, evidence for a direct relationship between Cd exposure and decreased bone H mass in the absence of tubular dysfunction has also been observed 14 .
To preserve bone health and provide suitable health promotion programs in Cd-exposed populations, the relationship between kidney dysfunction and bone metabolism needs to be elucidated. We aimed to quantify the association between renal abnormality and bone fracture risk in a Cdexposed population from Thailand using biomarkers.

Population and biological sampling
Study participants from Mae Sot, aged >50 yr with urinary cadmium levels quantified as higher than the reference level 5 µg/g Cr in a 2004 survey were enrolled 6 . The target sample size was 554 subjects, and of these 554 subjects, 419 (75.63 %) agreed to participate including 158 men and 261 women.
Informed consent was obtained from all participants. The study protocol was conducted in accordance with the Declaration of Helsinki as described in our previous report 15 .
Urine samples were collected in polyethylene bottles after the subjects underwent a physical examination and anthropometric measurements. Each urine sample was divided into three (3-5 ml) aliquots. In samples with pH <5, the pH of one of the three aliquots was adjusted to pH 6-8 by 0.5 N sodium hydroxide to prevent the degradation of β2microglobulin in acidic conditions. A trained nurse drew fiveto-ten milliliters of venipuncture blood. All aliquots were then frozen and stored at −20 °C until analysis.

Urinary cadmium measurement
Urinary cadmium concentrations were quantified using a flameless atomic-absorption spectrometer (Shimadzu Model AAS-6300, Japan), with palladium chloride in 5% nitric acid solution as a modifier. Method validation of the analytical techniques was performed and verified by certified standard reference materials (The National Institute of Standards and Technology, Washington, DC, USA) 16 . Urinary creatinine concentrations were measured by a method based on the Jaffe reaction.

Serum and urinary calcium measurements
Serum and urinary calcium were quantified by a colorimetric assay using an automated analyzer (Coulter HmX, Konelab 30 and Bechman Synchron CX3) at Mae Sot General Hospital. The laboratory was evaluated and certified by the Bureau of Laboratory Quality Standards, Ministry of Public Health, Thailand. The fractional excretion of calcium (FECa) was calculated based on the serum and urinary calcium concentrations 17 .

Renal and bone markers determination
The concentration of β2-microglobulin (β2-MG) in urine was determined by enzyme immunoassay (GLAZYME β2microglobulin-EIA test kit, Sanyo Chemical Industries, Ltd., Japan). Serum cystatin C concentrations (Cystatin C) were determined by a latex particle-enhanced turbidimetric immunoassay PET kit (Dako, Glostrup, Denmark). Serum osteocalcin (OC) was measured by immunoassay. Urinary type I collagen crosslinked N-telopeptide (NTx), was measured by a competitive enzyme immunoassay 15 . OC and NTx were used as biomarkers of bone turnover 15 .
Bone fracture risk was identified by a high NTx level. The cut-off values to indicate bone fracture risk were >66.2 nmol BCE/mmol Cr in men, and >89.0 nmol BCE/mmol Cr in women 11 .

Data analysis
U-Cd, β2-MG, and NTx were log-transformed to correct for departures from normal distributions. Bivariate associations were calculated using partial correlations, controlling for age, and visualized using scatter plots. Mean comparisons of biomarkers between genders were performed by ANCOVA adjusting for age. The Chi-square test was used to determine the distribution of subjects at high bone fracture risk according to categorized renal biomarker concentrations. Adjusted odds ratios were calculated by logistic regression. In the logistic regression model, the dependent variable was bone fracture risk indicated by high NTx level whereas the independent variables were age, BMI, smoking status (0=nonsmoker, 1=smoker), urinary cadmium, GFR, Cystatin C, β2-MG, and FECa. P-values of 0.05 or less were considered statistically significant.

Results
The mean age was 64.56 yr in men and 61.52 yr in women. After adjustment by mean age, the mean Cd exposure level was 6.81 µg/g Cr in men and 7.29 µg/g Cr in women, which was not significant difference between genders ( Table 1). The β2-MG mean concentration in men was higher than women were (765.60 vs 250.03 µg/g Cr, P<0.001). The Cystatin C mean concentration in men was borderline significantly higher than women were (1.36 vs 1.27, P=0.051). OC and NTx mean in women were significantly higher than men were. Partial correlations between OC and NTx and U-Cd, GFR, Cystatin C, and FECa are presented in Table 2. In men, OC was significantly correlated with BMI, GFR, Cystatin C, and β2-MG. NTx was significantly correlated with BMI, U-Cd, GFR, β2-MG, and FECa. In women, OC was significantly correlated with BMI, U-Cd, GFR, Cystatin C, β2-MG, and FECa. NTx was significantly correlated with BMI, U-Cd, GFR, β2-MG, and FECa. Scatter plots highlight significant positive correlations between NTx and U-Cd ( Figure 1) and NTx and FECa (Figure 2) in both genders.  In men, the prevalence of high bone fracture risk in individuals with β2-MG >1000 µg/g Cr was significantly higher compared to individuals with β2-MG <1000 µg/g Cr (29.73% vs 13.10%, P=0.010) ( Table 3). Additionally, the prevalence of high bone fracture risk in men with FECa >2% was significantly elevated relative to those with FECa<2% (54.55 vs 15.44%, P<0.001) ( Table 3). The prevalence of high bone fracture risk was significantly higher in women with FECa>2% relative to those with FECa<2% (56.10% vs 24.55%, P <0.001). We used logistic regression models, stratified by gender, to quantify the association between kidney dysfunction and high bone fracture risk. The dependent variable in the models was bone fracture risk indicated by excess NTx (>66.2 nmol BCE/mmol Cr in men, and >89.0 nmol BCE/mmol Cr in women), while age, BMI, U-Cd, GFR, Cystatin C, β2-MG, and FECa were modeled as the independent variables. Men and women with FECa>2% had odds of high bone fracture risk 6.03 and 3.29 times higher respectively, (Table 4) compared to gender-matched subjects with FECa<2%.

Discussion
Cadmium contamination in the Mae Sot region of Thailand has been reported since 2001 22 , and the exposed population has a high prevalence of proximal tubular dysfunction 5 . Cd disturbs bone metabolism via enhanced proximal tubular dysfunction 13 . An increased prevalence of osteoporosis in Cdexposed populations with kidney dysfunction has been reported 23 , however, glomerular dysfunction showed no association with reduced bone mass 24 . The proposed mechanisms of Cd osteotoxicity included Cd accelerated bone resorption 12 , inhibited incorporation of Ca ions into bone tissue, reduced Ca reabsorption from GI tract 25 , enhanced wasting of Ca into urine 26 , and decreased production of the active vitamin D metabolite, 1α,25(OH)2D 27 . In this study, we used published biomarker reference levels to define renal pathology and bone metabolism imbalance. Glomerular dysfunction was indicated by GFR <60 ml/min/1.73 m 2 body surface and cystatin C >1.4 mg/L. Cystatin C is recognized as a more accurate marker of glomerular dysfunction than serum creatinine 28 . Proximal tubular dysfunction was indicated by β2-MG >1000 µg/g Cr, a concentration that reflects irreversible proximal tubular dysfunction 20 . Ca wasting was indicated by FECa >2 % 21 . Urinary NTx, a bone resorption marker, is measured to show the risk of bone fracture risk (cut-off value; men >66.2, and women >89.0 nmol BCE/mmol Cr).

Glomerular
dysfunction and bone metabolism abnormalities were observed in chronic kidney disease patients. Specifically, reduced bone mass was associated with reduced glomerular filtration rate 21 . We identified a significant correlation between GFR and OC and NTx in both men and women (Table 2), indicating an association between glomerular dysfunction and bone remodeling. Serum cystatin C was also significantly correlated with OC in both genders. However, after classifying subjects according to bone fracture risk group and GFR or Cystatin C groups, no significant relationship between bone fracture risk and glomerular dysfunction was identified for either gender (Table 4). This result was in accordance with the report that the prevalence of osteoporosis did not relate to glomerular dysfunction 29 .
The proximal tubule is an accumulation site of Cd and a key target site for Cd toxicity 30 . Proximal tubular dysfunction occurs at an early stage of Cd intoxication 31 . β2-MG is a low molecular weight protein reabsorbed by the proximal tubules. An elevated urinary β2-MG indicates proximal tubular dysfunction. β2-MG has been recommended as a sensitive marker for Cd toxicity 32 . We found a positive correlation between β2-MG, OC, and NTx in both genders (Table 2). However, in logistic regression models, irreversible proximal tubular dysfunction did not relate to bone fracture risk (Table  4).
FECa levels greater than 2% indicate an impaired ability of the kidneys to reabsorb calcium back into the blood, which increases calcium wasting in the urine 16 . Increased calcium wasting was previously proposed as a sign of tubular damage and bone metabolism defects 33 . Subjects with increased calcium wasting have a higher risk of osteoporosis 34 . Calcium wasting into urine decreases serum calcium levels, causing the body to increase bone resorption to raise serum calcium 35 . Figure 2 shows a positive correlation between FECa and NTx, a bone resorption marker, which supports this assumption. High levels of bone resorption are normally balanced by increasing bone formation 35 , leading to high circulating levels of bone remodeling biomarkers. Here, we found a positive correlation between OC and FECa ( Table 2). This result agrees with the finding that Itai-itai patients who showed increased Ca wasting and high rates of bone formation had low bone mineralization 1 .
When we quantified the relationship between biomarkers of kidney dysfunction and bone markers, FECa was a significant explanatory factor of bone fracture risk in both men and women (Table 4). FECa >2% increased bone fracture risk 6.03-fold in men and 3.29-fold in women. In contrast, GFR <60 ml/min/1.73m 2 , Cystatin C >1.4 mg/l, and β2-MG >1000 µg/g Cr showed no significant relation to bone fracture risk. Ca wasting contributes to bone fracture risk more than proximal tubular dysfunction and glomerular dysfunction.
The presence of Ca wasting is a key risk factor for Cdinduced abnormal bone remodeling. Serum Ca and urinary Ca are commonly used to determine the kidneys Ca control. However, serum Ca is normally tightly regulated in narrow range and urinary Ca is easily affected by dietary Ca 17 . Thus, serum and urinary Ca do not specifically indicate the Ca controlling function of kidney. FECa has significant advantages over both of these methods. FECa is used to indicate the percentage of Ca filtered at the glomerulus not reabsorbed in the tubules, a marker unaffected by dietary Ca. FECa has been proposed as a marker to indicate the early stages of renal dysfunction 36 and is also inexpensive to measure relative to Cystatin C and β2-MG. A linear relationship between FECa and reduced bone mass, provided further evidence for the utility of FECa as a biomarker in this context 36 .
Women were previously proposed to be at higher risk for Cd osteotoxicity than men 37 but in this study men with high FECa had a bone fracture risk odds of 6.03 compared to low FECa men (P <0.001, Table 4). Evidence supporting a relationship between Cd exposure and elevated bone toxicity in men 38 . Male rats with heavy Cd exposure showed low levels of bone mineralization, decreased bone mass, and high levels of bone remodeling markers relative to non-exposed rats 25 . Men exposed to low levels of Cd also showed an inverse correlation between Cd exposure and bone mass 39 . Therefore, the bone health of Cd-exposed men should be monitored in addition to that of women.

Conclusion
The half-life of Cd in the human body is more than 10 years 25 . Cd related pathology appears in the elderly when the body cannot handle Cd toxicity. Since the Cd, contamination in the Mae Sot region of Thailand has not been fully remediated and further exposure continues, the inhabitants in this area continue to be at elevated risk of bone disease. Here, we found high bone fracture risk in study participants who showed FECa>2%. Calcium wasting was a dominant explanatory factor of bone fracture risk, showing the utility of monitoring this biomarker in Cd-exposed populations. Calcium supplements, renal toxicant exposure reduction, and regular health check-ups are essential preventive measures to reduce bone fracture risk in Cd-exposed populations. We recommended the use of FECa as an indicator of calcium wasting in Cd-exposed populations.